Lorentz force in water: evidence that hydronium cyclotron resonance enhances polymorphism.

There is an ongoing question regarding the structure forming capabilities of water at ambient temperatures. To probe for different structures, we studied effects in pure water following magnetic field exposures corresponding to the ion cyclotron resonance of H3O(+). Included were measurements of conductivity and pH. We find that under ion cyclotron resonance (ICR) stimulation, water undergoes a transition to a form that is hydroxonium-like, with the subsequent emission of a transient 48.5 Hz magnetic signal, in the absence of any other measurable field. Our results indicate that hydronium resonance stimulation alters the structure of water, enhancing the concentration of EZ-water. These results are not only consistent with Del Giudice's model of electromagnetically coherent domains, but they can also be interpreted to show that these domains exist in quantized spin states.

Lifestyle-related factors and environmental agents causing cancer: an overview.

The increasing incidence of a variety of cancers after the Second World War confronts scientists with the question of their origin. In Western countries, expansion and ageing of the population as well as progress in cancer detection using new diagnostic and screening tests cannot fully account for the observed growing incidence of cancer. Our hypothesis is that environmental factors play a more important role in cancer genesis than it is usually agreed. (1) Over the last 2-3 decades, alcohol consumption and tobacco smoking in men have significantly decreased in Western Europe and North America. (2) Obesity is increasing in many countries, but the growing incidence of cancer also concerns cancers not related to obesity nor to other known lifestyle-related factors. (3) There is evidence that the environment has changed over the time period preceding the recent rise in cancer incidence, and that this change, still continuing, included the accumulation of many new carcinogenic factors in the environment. (4) Genetic susceptibility to cancer due to genetic polymorphism cannot have changed over one generation and actually favours the role of exogenous factors through gene-environment interactions. (5) Age is not the unique factor to be considered since the rising incidence of cancers is seen across all age categories, including children, and adolescents. (6) The fetus is specifically vulnerable to exogenous factors. A fetal exposure during a critical time window may explain why current epidemiological studies may still be negative in adults. We therefore propose that the involuntary exposure to many carcinogens in the environment, including microorganisms (viruses, bacteria and parasites), radiations (radioactivity, UV and pulsed electromagnetic fields) and many xenochemicals, may account for the recent growing incidence of cancer and therefore that the risk attributable to environmental carcinogen may be far higher than it is usually agreed. Of major concern are: outdoor air pollution by carbon particles associated with polycyclic aromatic hydrocarbons; indoor air pollution by environmental tobacco smoke, formaldehyde and volatile organic compounds such as benzene and 1,3 butadiene, which may particularly affect children and food contamination by food additives and by carcinogenic contaminants such as nitrates, pesticides, dioxins and other organochlorines. In addition, carcinogenic metals and metalloids, pharmaceutical medicines and some ingredients and contaminants in cosmetics may be involved. Although the risk fraction attributable to environmental factors is still unknown, this long list of carcinogenic and especially mutagenic factors supports our working hypothesis according to which numerous cancers may in fact be caused by the recent modification of our environment.

The multitude and diversity of environmental carcinogens.

We have recently proposed that lifestyle-related factors, screening and aging cannot fully account for the present overall growing incidence of cancer. In order to propose the concept that in addition to lifestyle related factors, exogenous environmental factors may play a more important role in carcinogenesis than it is expected, and may therefore account for the growing incidence of cancer, we overview herein environmental factors, rated as certainly or potentially carcinogenic by the International Agency for Research on Cancer (IARC). We thus analyze the carcinogenic effect of microorganisms (including viruses), radiations (including radioactivity, UV and pulsed electromagnetic fields) and xenochemicals. Chemicals related to environmental pollution appear to be of critical importance, since they can induce occupational cancers as well as other cancers. Of major concerns are: outdoor air pollution by carbon particles associated with polycyclic aromatic hydrocarbons; indoor air pollution by environmental tobacco smoke, formaldehyde and volatile organic compounds such as benzene and 1,3 butadiene, which may particularly affect children, and food pollution by food additives and by carcinogenic contaminants such as nitrates, pesticides, dioxins and other organochlorines. In addition, carcinogenic metals and metalloids, pharmaceutical medicines and cosmetics may be involved. Although the risk fraction attributable to environmental factors is still unknown, this long list of carcinogenic and especially mutagenic factors supports our working hypothesis according to which numerous cancers may in fact be caused by the recent modification of our environment.

HIV-1 genome nuclear import is mediated by a central DNA flap.

HIV-1 and other lentiviruses have the unique property among retroviruses to replicate in nondividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV-1 reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap: the central DNA flap. We show here that the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import. Wild-type viral linear DNA is almost entirely imported into the nucleus where it integrates or circularizes. In contrast, mutant viral DNA, which lacks the DNA flap, accumulates in infected cells as unintegrated linear DNA, at the vicinity of the nuclear membrane. Consistently, HIV-1 vectors devoid of DNA flap exhibit a strong defect of nuclear import, which can be corrected to wild-type levels by reinsertion of the DNA flap sequence.

Specificity of anti-Nef antibodies produced in mice immunized with DNA encoding the HIV-1 nef gene product.

Mice immunized with plasmid DNA encoding Nef regulatory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies. After 4 intramuscular injections of 100 microg plasmid DNA, anti-Nef antibodies reached titers up to 2 x 10(4). A significant specific antibody response was maintained for at least 16 months. Using a set of seven 31-66 mer synthetic peptides covering the entire sequence of Nef, we analysed the specificity of ant-Nef antibodies. Interestingly, specific antibodies produced in response to Nef expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide (aa 141-205) for 3 of the 10 sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. Only 3 of the 5 Nef positive sera reacted with the C-terminal peptide. This suggests that specific antibodies induced by plasmid DNA as well as by the non-denatured protein recognize conformation-dependent epitopes. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity pattern. Interestingly, the analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed that plasmid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies.

Role of Mycoplasma penetrans endonuclease P40 as a potential pathogenic determinant.

Recently, we reported the purification to homogeneity and characterization of Ca(2+)- and Mg(2+)-dependent endonuclease P40 produced by Mycoplasma penetrans (M. Bendjennat, A. Blanchard, M. Loutfi, L. Montagnier, and E. Bahraoui, J. Bacteriol. 179; 2210-2220, 1997), a mycoplasma which was isolated for the first time from the urine of human immunodeficiency virus-infected patients. To evaluate how this nuclease could interact with host cells, we tested its effect on CEM and Molt-4 lymphocytic cell lines and on peripheral blood mononuclear cells. We observed that 10(-7) to 10(-9) M P40 is able to mediate a cytotoxic effect. We found that 100% of cells were killed after 24 h of incubation with 10(-7) M P40 while only 40% cytotoxicity was obtained after 72 h of incubation with 10(-9) M P40. Phase-contrast microscopy observations of P40-treated cells revealed morphological changes, including pronounced blebbing of the plasma membrane and cytoplasmic shrinkage characteristic of programmed cell death, which is in agreement with the internucleosomal fragmentation of P40-treated cell DNA as shown by agarose gel electrophoresis. We showed that (125)I-radiolabeled or fluorescein isothiocyanate-labeled P40 was able to bind specifically in a dose-dependent manner to the cell membrane of CEM cells, which suggested that the cytotoxicity of P40 endonuclease was mediated by its interaction with the cell surface receptor(s). The concentration of unlabeled P40 required to inhibit by 50% the formation of (125)I-P40-CEM complexes was about 3 x 10(-9) M, indicating a high-affinity interaction. Both P40 interaction and cytotoxicity are Ca(2+) dependent. Our results suggest that the cytotoxicity of M. penetrans observed in vitro is mediated at least partially by secreted P40, which, after interaction with host cells, can induce an apoptosis-like death. These results strongly suggest a major role of mycoplasmal nucleases as potential pathogenic determinants.

Viral load and neuropathology in the SIV model.

To investigate neuropathological processes involved in HIV infection, a longitudinal analysis of central nervous system (CNS) changes was performed using the SIV-infected macaque model. Five animals were studied during the early phase and 13 during the asymptomatic and symptomatic phases. Histopathological analyses were performed on one cerebral fixed hemisphere whereas on the other frozen hemisphere in situ hybridisation, immunohistochemistry and RT-PCR were performed. Viral load was quantified by in situ hybridisation, CD4 and CD8 T cell infiltration by immunohistochemistry and mRNA cytokine expression (IL1beta, IL2, IL6, TNFalpha, IFNgamma and TGF-beta1) by semiquantitative RT-PCR. As reported for HIV-infected humans, the neuropathological analysis of SIV infected animals revealed four distinct lesion profiles: minimal changes, early encephalitis, leukoencephalopathy and encephalitis. No relationship was found between neuropathological findings, numbers of SIV replicating cells and T cell infiltration. CNS infection was found to be an early event characterised by glial activation, an increase in the level of IL1beta, TNFalpha and IL6 mRNA expression. During the asymptomatic and symptomatic phases, IL6 and IL1beta mRNAs increase coincided with gliosis and the development of myelin lesions. The absence of relationship between neuropathological findings and viral load suggests that cerebral lesions are caused by an indirect mechanism. Inflammatory cytokine pattern associated with severe lesions show the key role of glial activation in the SIV neuropathological process.

Phase variations of the Mycoplasma penetrans main surface lipoprotein increase antigenic diversity.

Mycoplasma penetrans is a recently identified mycoplasma, isolated from urine samples collected from human immunodeficiency virus (HIV)-infected patients. Its presence is significantly associated with HIV infection. The major antigen recognized during natural and experimental infections is an abundant P35 lipoprotein which, upon extraction, segregates in the Triton X-114 detergent phase and is the basis of M. penetrans-specific serological assays. We report here that the P35 antigen undergoes spontaneous and reversible phase variation at high frequency, leading to heterogeneous populations of mycoplasmas, even when derived from a clonal lineage. This variation was found to be determined at the transcription level, and although this property is not unique among the members of the class Mollicutes, the mechanism by which it occurs in M. penetrans differs from those previously described for other Mycoplasma species. Indeed, the P35 phase variation was due neither to a p35 gene rearrangement nor to point mutations within the gene itself or its promoter. The P35 phase variation in the different variants obtained was concomitant with modifications in the pattern of other expressed lipoproteins, probably due to regulated expression of selected members of a gene family which was found to potentially encode similar lipoproteins. M. penetrans variants could be selected on the basis of their lack of colony immunoreactivity with a polyclonal antiserum against a Triton X-114 extract, strongly suggesting that the mechanisms involved in altering surface antigen expression might allow evasion of the humoral immune response of the infected host.

Programmed cell death as a mechanism of CD4 and CD8 T cell deletion in AIDS. Molecular control and effect of highly active anti-retroviral therapy.

Infection with human immunodeficiency virus (HIV) results in the progressive destruction of CD4 T lymphocytes, generally associated with progression of the disease. The progressive disappearance of CD4 T lymphocytes leads to the lack of control of HIV replication and to the development of severe immune deficiency responsible for the occurrence of opportunistic infections associated with AIDS. In this review we discuss premature lymphocyte apoptosis in the context of HIV infection as the consequence of the continuous production of viral proteins, leading to an unbalanced immune activation and to the triggering of apoptotic programs. The chronic immune activation induces the continuous expression of death factors which could turn lymphocytes, including CD4 T cells, CD8 CTL or APC, into effectors of apoptosis, leading to the destruction of healthy activated non-infected cells. Thus, programmed cell death would significantly contribute to peripheral T cell depletion in AIDS, particularly if the Th cell renewal is impaired. Under potent anti-retroviral therapies, a complete normalization of lymphocyte apoptosis is observed, concomitant with a partial restoration of the number and the functions of the immune system.

Characterization of humoral immune responses induced by immunization with plasmid DNA expressing HIV-1 Nef accessory protein.

Mice immunized with plasmid DNA encoding Nef accessory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies which were maintained for at least 16 months. These antibodies produced in response to Nef-expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide for three of the ten sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity pattern. Interestingly, the analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed that plasmid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies.

A longitudinal study of seroreactivity against Mycoplasma penetrans in HIV-infected homosexual men: association with disease progression.

We investigated the relationships between a putative cofactor of HIV infection, Mycoplasma penetrans, and the evolution of HIV disease. The evolution of titers of anti-M. penetrans antibodies in 58 randomly selected HIV-seropositive adult homosexual men was investigated. The median length of follow-up was 38 months. Thirty-six individuals was investigated. The median length of follow-up was 38 months. Thirty-six individuals (62.1%) remained M. penetrans seronegative (group 0). Fourteen patients (24.1%) had consistently low antibody titers or low antibody titer(s) in at least one sample and negative test(s) in the other(s). This pattern was possibly associated with latent or earlier infection (group 1). Eight patients (13.8%) had moderate to high antibody titers for long periods, indicating an active and persistent M. penetrans infection (group 2); four patients in this group presented a serological reactivation and thus probably developed an acute infection during the study; two had a stable and moderate level of antibody throughout the study; in two patients the antibody titers decreased substantially. Interestingly, CD4 cell counts declined more rapidly in group 2 than in group 0 (medians of -4.5 versus -2.1 cells/mm3/month, p < 0.05 and -0.16 versus 0 cell percentage/month, p < 0.05), whereas there was no significant difference between groups 1 and 0 (medians of -2.0 versus -2.1 cells/mm3/month and -0.15 versus 0 cell percentage/month). In patients with serological reactivation, the viral load was higher in sera with higher M. penetrans antibody titers. These findings suggest an association between active M. penetrans infection and progression of HIV disease.

Molecular evidence for nosocomial transmission of human immunodeficiency virus from a surgeon to one of his patients.

We have investigated the molecular evidence in favor of the transmission of human immunodeficiency virus (HIV) from an HIV-infected surgeon to one of his patients. After PCR amplification, the env and gag sequences from the viral genome were cloned and sequenced. Phylogenetic analysis revealed that the viral sequences derived from the surgeon and his patient are closely related, which strongly suggests that nosocomial transmission occurred. In addition, these viral sequences belong to group M of HIV type 1 but are divergent from the reference sequences of the known subtypes.

Simian immunodeficiency virus replicates to high levels in sooty mangabeys without inducing disease.

A serologic survey of primates living in a French zoo allowed identification of three cases of infection with simian immunodeficiency virus in sooty mangabeys (Cercocebus atys) (SIVsm). Viral isolates, which were designated SIVsmFr66, SIVsmFr74, and SIVsmFr85, were obtained after short-term culture of mangabey lymphoid cells. Phylogenetic analysis of gag and env sequences amplified directly from mangabey tissues showed that the three SIVsmFr were genetically close and that they constituted a new subtype within the diverse SIVsm-SIVmac-human immunodeficiency virus type 2 (HIV-2) group. We could reconstruct the transmission events that likely occurred in 1986 between the three animals and evaluate the divergence of SIVsmFr sequences since transmission. The estimated rate of mutation fixation was 6 x 10(-3) substitutions per site per year, which was as high as the rate found for SIVmac infection in macaques. These data indicated that SIVsmFr replicated at a high rate in mangabeys, despite the nonpathogenic character of infection in this host. The viral load evaluated by competitive PCR reached 20,000 viral DNA copies per 10(6) lymph node cells. In addition, productively infected cells were readily detected in mangabey lymphoid tissues by in situ hybridization. The amounts of viral RNA in plasma ranged from 10(5) to 10(7) copies per ml. The cell-associated and plasma viral loads were as high as those seen in susceptible hosts (humans or macaques) during the asymptomatic stage of HIV or SIVmac infections. Thus, the lack of pathogenicity of SIVsm for its natural host cannot be explained by limited viral replication or by tight containment of viral production.

HIV-1-resistance phenotype conferred by combination of two separate inherited mutations of CCR5 gene.

BACKGROUND: Despite multiple exposures to HIV-1, some individuals remain uninfected, and their peripheral-blood mononuclear cells (PBMC) are resistant to in-vitro infection by primary HIV-1 isolates. Such resistance has been associated with a homozygous 32-base-pair deletion (delta 32) in the C-C chemokine receptor gene CCR5. We examined other mutations of the CCR5 gene that could be associated with resistance to HIV-1 infection. METHODS: We assessed the susceptibility of PBMC to in-vitro infection by HIV-1 isolates that use the CCR5 as the major coreceptor for viral entry in 18 men who had frequent unprotected sexual intercourse with a seropositive partner. We also did genotypic analysis of CCR5 alleles. One of the 18 exposed but uninfected men (who we refer to as ExU2) showed total resistance to in-vitro infection by CCR5-dependent viruses, and was found to carry a CCR5 delta 32 allele and a single point mutation (T-->A) at position 303 on the other allele. To find out whether the CCR5 mutation was restricted to ExU2's family or existed in the general population, we did genetic analyses of the CCR5 genotype in ExU2's father and sister and also in 209 healthy blood donors who were not exposed to HIV-1. FINDINGS: The m303 mutation found in ExU2 introduced a premature stop codon and prevented the expression of a functional coreceptor. The family studies revealed that the m303 mutant allele was inherited as a single mendelian trait. Genotype analysis showed that three of the 209 healthy blood donors were heterozygous for the mutant allele. INTERPRETATION: We characterise a new CCR5 gene mutation, present in the general population, that prevents expression of functional coreceptors from the abnormal allele and confers resistance to HIV-1 infection when associated to the delta 32 CCR5 mutant gene.

Efficacy and safety of Buxus sempervirens L. preparations (SPV(30)) in HIV-infected asymptomatic patients: a multicentre, randomized, double-blind, placebo-controlled trial.

The objective of the present study was to compare the efficacy and safety of two doses of SPV(30) in HIV asymptomatic patients. The study was designed as a randomized double-blind multicentre trial of two doses of SPV(30) (990 mg/d and 1980 mg/d) versus placebo. 145 previously untreated subjects with asymptomatic HIV infection (CDC group IV) and CD4 cell counts between 250 and 500 × 10(6)/1 were recruited. There was a statistically significant difference in therapeutic failures between groups in favor of SPV(30) 990 mg including decreases of CD4 cell count < 200 × 10(6)/1 and/or number of clinical aggravations (progression to AIDS or AIDS related complex). The treatment groups differed statistically in the rate of disease progression also in favor of SPV(30) 990 mg/d. Fewer patients receiving SPV(30) 990 mg/d had at the end an increase of viral load greater than 0.5 log (P = 0.029). No severe side-effects were reported in the 3 groups. From these results we conclude that SPV(30) 990 mg/d has beneficial effects in HIV asymptomatic patients and appears to delay the progression of HIV disease.

Spiralin, a mycoplasmal membrane lipoprotein, induces T-cell-independent B-cell blastogenesis and secretion of proinflammatory cytokines.

Mycoplasmas are bacteria which can cause respiratory, arthritic, and urogenital diseases. During the early phase of infection, mycoplasmas usually induce an inflammatory response and a humoral response preferentially directed against their membrane-bound, surface-exposed lipoproteins. In this report, we describe the effects on immune cells of spiralin, a well-characterized mycoplasmal lipoprotein. Purified spiralin stimulated the in vitro proliferation of human peripheral blood mononuclear cells and murine splenocytes. The stimulation pathway was probably different from that followed by Escherichia coli lipopolysaccharide because the effect of spiralin was not abolished by polymyxin B. Comparison of the effects of whole, native spiralin with those induced by proteinase K-digested spiralin or by the C-terminal half of spiralin (peptide p[13.5]T) revealed that the first half of the protein, which contains the lipoylated N terminus, is responsible for the mitogenic activity. In contrast to whole spiralin, proteinase K-digested spiralin did not trigger murine B-cell differentiation and immunoglobulin G and M secretion. Stimulation of human or murine immune cells led to early secretion of proinflammatory cytokines (human tumor necrosis factor alpha and murine interleukin 1 or 6). Spiralin induced the T-cell-independent blastogenesis of murine B cells but did not stimulate T cells. Altogether, our data demonstrate that spiralin possesses potent immunostimulating activity, similar to that reported for lipoproteins of pathogenic gracilicutes (gram-negative eubacteria; e.g., Borrelia burgdorferi OspA and E. coli Braun lipoprotein), and are consistent with the fact that lipoproteins are major antigens during mycoplasma infections.

Oxidative protein damage and degradation in lymphocytes from patients infected with human immunodeficiency virus.

It has been proposed that oxidative stress is the common mediator of apoptotic cell death in AIDS. However, mechanistic relationships between oxidative damage and cell death are far from clear. It is reported here that the mitogenic activation of T lymphocytes from human immunodeficiency virus-positive subjects involves perturbation of redox balance, as indicated by the increase in hydroethydine intracellular oxidation and manganese superoxide dismutase adaptive induction. Principal molecular targets of oxidative injury are cellular proteins whose content in carbonyl groups increases together with a dramatic increase in degradation of newly synthesized proteins catalyzed by the ATP- and ubiquitin-dependent proteolytic system. The major consequence of this metabolic anomaly is the decrease in protein cell mass leading to cells that are smaller than normal at lethal mitosis.